KR20170087835A - A Novel Specific Monoclonal Antibody for acetylated mouse BubR1 and preparation method of the same - Google Patents

Abstract

The present invention provides a rabbit immuno-monoclonal antibody against acetylated mouse BubR1 and a method for its preparation,
Further, the above monoclonal antibody can be used to measure the degree of cell division checkpoint activity on the basis of the degree of acetylation of BubR1, to detect tumor diseases based on cell division abnormality, cancer diagnosis, anticancer screening method, or cell division cycle control method do.

Description

[0001] The present invention relates to a novel monoclonal antibody against acetylated mouse BubR1 and a preparation method thereof,

The present invention relates to a specific rabbit immunoglobulin monoclonal antibody against acetylated mouse BubR1 and a preparation method thereof, and more particularly to a hybridoma cell producing the above-mentioned specific monoclonal antibody and the monoclonal antibody, The antibody may provide a measure of cell division checkpoint activity on the basis of the degree of acetylation of BubR1, detection of a disease based on cell division abnormality, cancer diagnosis, anticancer screening method, or cell division cycle regulating method.

The most important factor in cell division is to accurately convey the same genetic information as the daughter cell to the daughter cell. In particular, the time of chromosome segregation, that is, the transition period from the middle stage to the late stage of cell division, is the apex of cell division, and the transfer of the complete genome to the daughter cell requires a check- Spindle Assembly Checkpoint (SAC) requires normal operation [Taylor SS, Scott MI, Holland AJ (2004). The spindle checkpoint: a quality control mechanism which ensures accurate chromosome segregation. Chromosome Res 12: 599-616]. Normally, cell division usually ends within 30 minutes, but if one chromosome does not adhere properly to the spindle, cell division will be delayed for up to 16 hours. The regulation of APC-Cdc20 by the spindle checkpoint (SAC) and the activated SAC inhibit the post-translational complex (APC / C) required for the post-apoptotic process [Yu H (2002). Curr Opin Cell Biol 14: 706-714]. APC / C destroys E3 ligase rosecurein and cyclin B, which are multimeric complexes [Yu, ibid]. If the SAC fails to function properly, the cell will die or produce a diploid diploid, which is known to be an important feature of cancer cells [Kops GJ, Weaver BA, Cleveland DW (2005) . Nat Rev Cancer 5: 773-785; Pihan G.A. et al (1999) Cancer Biology 9: 289-302). In addition, a failure of SAC can lead to a chromosomal instability cancer (CIN) that is progressive and difficult to treat [Hannahan D et al, Cell 100: 57-70 (2000); Li R et al. PNAS 97: 3236-3241 (2000). Since cancer cells divide unlimitedly, controlling cell division is a shortcut to cancer therapy. For this, it is necessary to examine the interactions of several factors at cell division checkpoints, the mechanism of their regulation, and the development of substances that can check the activity of these checkpoints. need. Of the factors that constitute the cell division checkpoint, SAC, BubR1 is known to be important for inhibiting APC / C by binding directly to APC / C [Yu, ibid]. However, the precise APC / C regulatory mechanism of this BubR1 protein and the antibody specifically recognizing it are unknown.

Furthermore, no antibodies known to detect the mouse acetyl BubR1 protein have been known to date. As an animal capable of in vivo experiment, a mouse model is representative, but it is impossible to carry out an experiment such as screening of an anticancer drug using a mouse as a conventional antibody. Therefore, development of an antibody capable of recognizing the mouse acetyl BubR1 protein is urgent.

In addition, monoclonal antibodies are antibodies recognizing one epitope. Therefore, the monoclonal antibody is low in cross-reactivity and can be produced semi-permanently for the purpose of identifying the presence of the epitope. Therefore, the monoclonal antibody can be stably supplied. In addition, since it has high specificity and high affinity, it is widely used not only for the purpose of detecting a specific protein in research but also as a therapeutic antibody (antibody medicine) and diagnostic tool for cancer. Rabbit immunoglobulin monoclonal antibodies have been used as monoclonal antibodies for research and diagnosis. Specifically, rabbit immunity antibodies have generally been reported to have higher affinity and higher specificity (Rossi, S et al., Am J Clin Pathol., 124 (2), 295-302 (2005); Rocha R et al., Pathol Res Pract. , 204 (9), 655-662 (2008); Tao, GZ et al, Exp Cell Res 312, 411-422 (2006)]. In particular, rabbits are known to produce antibodies against antigens with greater immunogenicity than mice and other animals [Raybould, TJ et al, Science 240 , 1788-1790 (1988): Feng, L et al, Am J Transl Res 3, 269-274 (2011)].

The present inventors have demonstrated that BubR1 acetylation acts as a molecular switch to control cell division checkpoint activity, suggesting that BubR1 acetylation can be used as a cell division checkpoint specific indicator [Choi et al., EMBO Journal , 28 (14), 2077-2089 (2009)). BubR1 acetylation site-inactivated BubR1-K243R / + mice embryonic fibroblasts (MEFs) were found to produce about 40% of cancers by themselves. BubR1 acetylation was found to be involved in the mobilization-spindle assembly of spleen and spindle assembly checkpoint (Park et al., J. Cell Biol ., 202 (2), 295-309 (2013)], which regulate the two most important cell division functions.

Korean Patent Laid-Open No. 10-2011-0019495 discloses a method for producing a polyclonal antibody against BubR1 derived from human, Mus musculus, Galus gallus, . However, no method of producing rabbit immuno-monoclonal antibodies of acetylated mouse BubR1 has been disclosed or suggested in any of the above references.

Accordingly, an object of the present invention is to provide a novel hybridoma cell line for rabbit-immunized acetylated mouse BubR1, a rabbit immuno-monoclonal antibody derived from the cell line, and a method for producing the same.

It is another object of the present invention to provide the use of said monoclonal antibody.

It is still another object of the present invention to provide a cancer diagnostic kit.

Other objects of the present invention can be found within the scope of the embodiments of the detailed description of the specification.

The above object of the present invention is achieved by a method for determining a target peptide antigen for an acetylated mouse BubR1 and producing a polyclonal hybridoma cell; The polyclonal hybridoma cells prepared in the above step were transfected with GST-tagged mouse BubR1 (1-514 amino acid) recombinant protein to prepare BubR1-specific polyclonal hybridoma mAb BubR1-SNU-20 cells No. KCLFR-BP-00390, deposit on Apr. 19, 2017); Selecting and culturing monoclonal hybridoma cells in the BubR1-specific polyclonal hybridoma mAb BubR1-SNU-20 cells obtained in the above step, and purifying the culture medium; Immunoprecipitation and Western blotting of HeLa cells were carried out to identify monoclonal antibodies specific for acetylated mouse BubR1 in the purified culture obtained in the above step to obtain monoclonal hybridoma cells mAcBubR1-SNU SN-20-7 (Korean Cell Line Bank, Accession No. KCLFR-BP-00392, deposited at the Korean Cell Line Bank, Accession No. KCLFR-BP-00391, 2017.01.19) and monoclonal hybridoma cells mAcBubR1-SNU-20-9 (Korean Cell Line Bank, Accession No. KCLFR-BP-00393, deposited at 2017.01.19) Clone antibody; Immunoprecipitation and Western blotting of mouse embryonic fibroblast were performed to identify monoclonal antibodies specific for endogenous acetylated mouse BubR1 in the monoclonal antibodies obtained in the above step, RTI ID = 0.0 > monoclonal < / RTI > antibody mAcBubR1-SNU-20-7.

The present invention provides a monoclonal antibody against rabbit-immunized acetylated mouse BubR1 which is superior in specificity and reactivity to a mouse monoclonal antibody and a method for producing the same. In addition, the present invention provides a monoclonal antibody against BubR1 There is an excellent effect of providing a method of measuring cell division checkpoint activity on the scale of acetylation degree, detecting a disease based on cell division abnormality, cancer diagnosis, screening method of cancer drug, or regulating cell division cycle.

1 is a schematic diagram of a novel GST-tagged mouse BubR1 (1-514 amino acid) recombinant protein according to the present invention.
FIG. 2 is a graph showing the relationship between the acetylated BubR1 specificity of the mouse acetylated BubR1 polyclonal hybridoma cell culture according to the present invention and the specificity of BubR1 containing the acetylated lysine residue This is a picture of a western blot showing superiority.
3 is a schematic diagram of Myc-tagged mouse BubR1 according to the present invention.
Figure 4 shows that the plasmid containing the Myc-tagged mouse BubR1 was transfected into HeLa cells and then transfected with the monoclonal antibodies mAcBubR1-SNU-20-4, mAcBubR1-SNU-20 -7 and mAcBubR1-SNU-20-9 after immunoprecipitation experiments.
Figure 5 shows the results of Western blotting after immunoprecipitation experiments of monoclonal antibody mAcBubR1-SNU-20-7, which specifically binds to mouse acetyl BubR1 protein using Mouse Embryonic Fibroblast (mouse embryonic fibroblast) according to the present invention It is a picture.

The present invention discloses monoclonal antibodies against acetylated mouse BubR1 and methods for their preparation.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Those skilled in the art will appreciate that those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. Techniques or techniques related to the present invention include [Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas. Generation of rabbit monoclonal antibodies (Methods Mol Biol., 2014; 1131: 71-9) and Generation of rabbit monoclonal antibodies (Proc Natl Acad Sci US A. 1995 92 methods Mol Biol, 2014; 1131: . 71-9)] it has been described in detail with respect to the bar in vitro and in vivo, and the antibody producing hybridoma obtained in the method described.

In the present invention, the term "antibody" is a molecule that specifically binds to an antigen and includes dimer, trimer and multimer, antibody, recombinant, processed and camelized antibody. In addition, the antibody may be in its complete form as well as in the form of a functional fragment of an antibody molecule. As used herein, the term "monoclonal antibody" refers to a single molecule composition of antibody molecules obtained in a substantially identical population of antibodies, which monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. "Hybridoma cell line" is a hybrid cell line obtained by fusing tumor cells and normal cells having any function, and is used for the production of monoclonal antibodies and the like by obtaining the cells having both the proliferation and the function of the living cell of the tumor cells Cell line.

The term " composition " in the present invention can be provided as a composition for preventing and treating cancers such as cattle, horses, sheep, pigs, chlorine, camels,

The term "prevent" used in the present invention means all actions in which the induction of cancer is suppressed or delayed by the administration of the composition comprising the antibody of the present invention, and "treatment" Or any change that beneficially modifies it.

When used as a therapeutic antibody, the antibody can be indirectly coupled (for example, covalently bonded) to an existing therapeutic agent directly or through a linker, and then introduced into the body in the form of antibody-therapeutic agent conjugate to be used for prevention or treatment of cancer have. Therapeutic agents that may be used include chemotherapeutic agents, immunotherapeutic agents, cytokines, chemokines, antiviral agents, biological agents, enzyme inhibitors and the like.

The term " composition comprising an antibody " in the present invention includes a pharmaceutically acceptable carrier and may be formulated for human or veterinary use. Pharmaceutical compositions for oral administration may be presented as discrete units such as capsules or tablets; Powders or granules; Syrups or suspensions (in aqueous or non-aqueous liquids; edible foams or whips; or emulsions). Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffering agents, bacteriostats and solutes (which are substantially contemporaneous with the blood of the recipient); And aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients that may be used in the injectable solution include, for example, water, alcohols, polyols, glycerin and vegetable oils. Such compositions may be presented in unit-dose (one-time) or multi-dose (several-dose) containers, such as sealed ampoules, and may be presented in a sterile liquid carrier, Lt; RTI ID = 0.0 > freeze-drying < / RTI > Immediate injectable solutions and suspensions may be prepared from sterile powders, granules and tablets.

A " kit " of the present invention includes not only antibodies of the present invention but also tools, reagents and the like commonly used in the art used for immunological analysis. Such tools / reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

In the present invention, the term "detection of acetylation of BubR1 protein" means qualitatively or quantitatively measuring the signal size of a detection label bound to an antigen-antibody conjugate to determine the presence and amount of acetylated BubR1 do. These detection kits include not only antibodies of the present invention but also tools, reagents and the like commonly used in the art used for immunological analysis. Such tools / reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

Immunoprecipitation Assay, Immunodiffusion Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, assay, complement fixation assay, FACS, protein chip, and the like.

Labels that enable qualitative or quantitative measurement of the formation of antigen-antibody conjugates include, but are not necessarily limited to, enzymes, minerals, ligands, emitters, microparticles, redox molecules, and radioisotopes . Enzymes that can be used as detection labels include, but are not limited to,? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, A gene coding for a gene coding for a gene encoding a β-catenin gene such as β-catenin, β-catenin, β-catenin, β-catenin, And the like. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthalate, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like.

Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Examples of the redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4W (CN) 8, [Os (bpy) RU (bpy) 3] 2+, [MO (CN) 8] 4-, and the like. Radiation isotopes include, but are not limited to, 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re.

The term " cross-reactivity " in the present invention is a phenomenon in which even a plurality of multiple antigenic determinants exist in an antigenic substance, so that antibodies against a specific antigenic determinant can also react with other similar antigenic determinants. The term " marking substance " is a substance that is labeled to detect a desired substance or phenomenon, and a fluorescent substance, a radioactive isotope, a ferritin, an enzyme, and the like are used. The labeling substance can be detected by fluorescence microscopy, radioautography, an optical microscope, an enzyme reaction, and the like, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to examples.

[Example 1] Crystallization of a target peptide antigen of the present invention and production of polyclonal hybridoma cells

Antibodies of the acetylated mouse BubR1 of the present invention were prepared using the information and sequences in Table 1 below. For the production of an efficient antibody, the left and right sequences were selected so that the lysine (K) residue was located in the middle, and the following antibody-producing target peptide sequence No. 1 was selected (Table 1).

mouse BubR1 accession number Q9Z1S0
Mouse BubR1 243rd lysine (K) residue surrounding sequence PISRVGGAL K APGQSR
Antibody production target Peptides  SEQ ID NO: 1 cGGAL (ac K) APGQS

P: Proline, G: Glycine, Q: Glutamine, S: Serine, K: Acetylation site)

The antibody-producing target peptide SEQ ID NO: 1 was injected into rabbits, and cells producing antibodies against the antigen were generated. Antibody-producing cells (lymphocytes, B cells) obtained from the rabbit spleen were transfected into myeloma cells (myeloma, 240E-W2, Abcam (R)). The fused cells were obtained by immunoassaying multiple cell types or multiclonal hybridoma cells through ELISA.

[Example 2] Preparation of GST-tagged mouse BubR1 (1-514 amino acid) recombinant protein of the present invention and selection of BubR1 specific polyclonal hybridoma mAbBubR1-SNU-20 cell

In order to select polyclonal hybridoma cells specifically recognizing only acetylated mouse BubRl through in vitro acetylation experiments of the polyclonal hybridoma cells obtained in Example 1, the GST- tagged mouse BubR1 (1-514 amino acid) recombinant protein.

The GST-tagged mouse BubR1 (1-514 amino acid) recombinant protein was added with Acetyl-CoA and divided into two groups. One group was added GST-P / CAF HAT domain, one group was excluded, (AC) and GST-tagged mouse BubR1 (1-514 amino acid) recombinant proteins were obtained by acetylating the recombinant proteins of the respective 'GST-tagged mouse BubR1 (1-514 amino acid) (NAC) 'were prepared. The polyclonal hybridoma cell culture broth was added to the above two groups in a blocking solution containing 5% defatted milk powder at a ratio of 1: 3, followed by electrophoresis and Western blotting.

As a result of Western blotting, the mAcBubR1-SNU-20 cells in the polyclonal hybridomas obtained in Example 1 were found to exhibit superiority as an antibody specifically recognizing BubR1 containing the acetylated lysine residue at residue 243 (Fig. 2).

The polyclonal hybridoma cells produced according to this example were designated mAcBubR1-SNU-20 and deposited under the Budapest Treaty with the Korean Depository No. KCLFR-BP-00390 at the International Depository Agency, Korea Cell Line Bank (Seoul, Korea) The deposit has been completed.

[Example 3] Selection of monoclonal hybridoma cells in BubR1-specific polyclonal hybridoma mAb BubR1-SNU-20 cells and purification of culture medium

After selecting single-line hybridoma cells (single line or subclone) in the polyclonal hybridoma mAb BubR1-SNU-20 cells selected in Example 2,

Additional selected monoclonal hybridoma cells were cultured in RPMI 1640 medium containing 10% FBS (fetal bovine serum), 1% penicillin and streptomycin at 37 ° C under 5% CO ₂ . Each of the monoclonal antibodies contained in the culture broth was further subjected to immunochemical precipitation through each HeLa cell or mouse embryonic fibroblast and Western blot experiment as in Examples 4 and 5 to specifically recognize the acetylated mouse BubR1 Were selected and identified as monoclonal antibodies.

[Example 4] Confirmation of specificity of monoclonal antibody against acetylated mouse BubR1 by immunoprecipitation of HeLa cells and Western blotting

HeLa cells transfected with Myc-tagged mouse BubR1 (FIG. 3) were treated with nocodazole (200 ng / ml), synchronized and gently patched in a cell culture container to remove medium cells mitotic shake off). The mid-stage cells were collected, and the cell extracts obtained therefrom were used to perform immunoprecipitation experiments with an anti-9E10 antibody. Then, the acetylated mouse BubR1 monoclonal antibody of Example 3 was treated with Western blot , It was confirmed that mAcBubR1-SNU-20-4, mAcBubR1-SNU-20-7 and mAcBubR1-SNU-20-9 specifically bind to each other and can be used as a monoclonal antibody (FIG. 4) .

The monoclonal hybridoma cells produced according to this example were named mAcBubR1-SNU-20-4, mAcBubR1-SNU-20-7 and mAcBubR1-SNU-20-9, (KCLFR-BP-00391, KCLFR-BP-00392 and KCLFR-BP-00393, respectively)

[Example 5] Identification of monoclonal antibody against acetylated mouse BubR1 by immunoprecipitation of mouse embryonic fibroblast and Western blotting

Endogenous mouse acetylated To identify antibodies recognizing BubR1, mouse embryonic fibroblasts were treated with nocodazole (400 ng / ml) (mitotic shake off). After collecting the above cells and performing immunoprecipitation experiments with the anti-BubR1 antibody using the cell extracts obtained therefrom, the acetylated mouse BubR1 monoclonal antibodies of Example 4 were treated with Western blotting As a result, it was confirmed that mAcBubR1-SNU-20-7 specifically binds to these and can be used as a monoclonal antibody (FIG. 5).

As described above, the present invention provides a monoclonal antibody against rabbit-immunized acetylated mouse BubR1, which is superior in specificity and reactivity to a mouse monoclonal antibody, and a method for producing the same. Therefore, By using the antibody, the degree of acetylation of BubR1 can be used as a measure to measure cell division checkpoint activity, to detect tumor diseases based on cell division abnormality, to diagnose cancer, to screen cancer drug, or to control cell division cycle, Which is a very useful invention in diagnosis and further cancer prevention and treatment industry.

<110> Seoul National University R & DB Foundation <120> A Novel Specific Monoclonal Antibody for Acetylated Mouse BubR1          and preparation method of the same <130> P5953 <150> KR 10-2016-0006851 <151> 2016-01-20 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 1 <220> <221> PEPTIDE <222> (1) <223> 6th amino acid Lysine is acetylated. <400> 1 Cys Gly Gly Ala Leu Lys Ala Pro Gly Gln Ser   1 5 10

Claims (14)

Determining a target peptide antigen for acetylated mouse BubR1 and producing polyclonal hybridoma cells; The polyclonal hybridoma cells prepared in the above step were cultured in a BubR1 specific polyclonal hybridoma mAb BubR1-SNU-20 cell (KCLFR-BP-00390) using GST-tagged mouse BubR1 (1-514 amino acid) ); Selecting a monoclonal hybridoma cell from the BubR1-specific polyclonal hybridoma mAb BubR1-SNU-20 cells selected in the above step, and culturing and then purifying the culture solution; Immunoprecipitation and Western blotting of HeLa cells were carried out to identify monoclonal antibodies specific for acetylated mouse BubR1 in the purified culture obtained in the above step to obtain monoclonal hybridoma cells mAcBubR1-SNU (KCLFR-BP-00391), mAcBubR1-SNU-20-7 (KCLFR-BP-00392) and mAcBubR1-SNU-20-9 To obtain a monoclonal antibody; Immunoprecipitation and Western blotting of mouse embryonic fibroblast were performed to identify monoclonal antibodies specific for endogenous acetylated mouse BubR1 in the monoclonal antibodies obtained in the above step, To obtain the monoclonal antibody mAcBubRl-SNU-20-7. &Lt; / RTI &gt;
2. The monoclonal antibody of claim 1, wherein the target peptide antigen for producing the monoclonal antibody comprises a heavy chain variable region consisting of the lysine residue of mouse BubRl acetylated SEQ ID NO: 1 (cGGAL (acK) APGQS) Way. A monoclonal antibody against rabbit-immunized acetylated mouse BubRl produced according to the method of claim 1 or 2. &lt; Desc / Clms Page number 36 &gt; 4. The antibody of claim 3, wherein said monoclonal antibody is derived from BubR1-specific polyclonal hybridoma cell line mAcBubR1-SNU-20 (KCLFR-BP-00390). 4. The monoclonal antibody of claim 3, wherein the monoclonal antibody is selected from the group consisting of rabbit-immunized hybridoma cell lines mAcBubR1-SNU 20-4 (KCLFR-BP-00391), mAcBubR1- SNU 20-7 (KCLFR- -9 (KCLFR-BP-00393). The monoclonal antibody of claim 5 does not exhibit cross-reactivity to the non-acetylated BubR1 protein and the BubR1 protein wherein the lysine residue is substituted with an arginine. 5. The antibody of claim 5, wherein the monoclonal antibody further comprises a labeling substance. A composition comprising the monoclonal antibody of claim 5. The composition of claim 8 is a pharmaceutical composition for preventing or treating cancer. A cancer diagnostic kit comprising the composition of claim 8. A method for detecting the degree of acetylation of a BubR1 protein comprising determining the degree of acetylation of the BubR1 protein using the monoclonal antibody of claim 5. A kit for detecting the acetylation degree of a BubR1 protein in a sample, which comprises the monoclonal antibody of claim 5. A polyclonal hybridoma cell line mAcBubR1-SNU-20 (KCLFR-BP-00390) producing the monoclonal antibody of claim 5. The monoclonal hybridoma cell line mAcBubR1-SNU 20-4 (KCLFR-BP-00391), mAcBubR1-SNU 20-7 (KCLFR-BP-00392), mAcBubR1-SNU 20-9 KCLFR-BP-00393).

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